Our long-term goal is to determine mechanisms by which the PI3K/AKT pathway, which is commonly activated in human cancers, increases expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-la (HIF-la). We also wish to understand what role this may play in the efficacy of EGFR inhibitors, which are currently being used in the clinic. VEGF, a key mediator of angiogenesis, is often overexpressed in human cancers. Hypoxia has long been recognized to be a potent inducer of VEGF expression through the transcription factor HIF-1. We have found that the PI3K/AKT pathway plays an important role in VEGF regulation through at least two different mechanisms. First, the PI3K/AKT pathway increases transcription by increasing binding of the transcription factor Sp1 to the VEGF proximal core promoter. Secondly, the AKT pathway can increase the expression of HIF-1a, which can also lead to increased VEGF transcription, particularly in hypoxia. In Specific Aim 1 we will explore the mechanisms by which AKT leads to increased Sp1-mediated transactivation of the VEGF promoter. In Specific Aim 2 we will study the potential role of glycogen synthase kinase-3p (GSK-3J3), a downstream target of AKT involved in protein translation, on increasing HIF-1 a. A number of drugs are currently being tested in the clinic that may work in part through the mechanisms described above. EGFR inhibitors (e.g. gefitinib, erlotinib) decrease PI3K/AKT signaling and we have found that these inhibitors also decrease HIF-1a and VEGF expression. Our preliminary data suggest that gefitinib may increase tumor oxygenation, which should lead to increased radiosensitization. Therefore, in Specific Aim 3 we will study the effects of EGFR inhibition on HIF-1 a and VEGF expression and on tumor oxygenation in vivo and the effects on radiosensitivity. LAY SUMMARY: We will study how the expression of VEGF, an important mediator of blood vessel growth, is increased in human tumors. The clinical importance of these studies is that EGFR inhibitors currently being used in the clinic may work though these pathways to increase the oxygenation of tumors. Increased oxygenation should make tumors more sensitive to radiation;therefore, our studies may be important in helping to optimize the combination of EGFR inhibitors with radiation.